Search

Acalabrutinib is a covalent Bruton tyrosine kinase (BTK) inhibitor approved for relapsed/refractory mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. A major metabolite of acalabrutinib (M27, ACP-5862) was observed in human plasma circulation. Subsequently, the metabolite was purified from an in vitro biosynthetic reaction and shown by nuclear magnetic resonance spectroscopy to be a pyrrolidine ring-opened ketone/amide. Synthesis confirmed its structure, and covalent inhibition of wild-type BTK was observed in a biochemical kinase assay. A twofold lower potency than acalabrutinib was observed but with similar high kinase selectivity. Like acalabrutinib, ACP-5862 was the most selective toward BTK relative to ibrutinib and zanubrutinib. Because of the potency, ACP-5862 covalent binding properties, and potential contribution to clinical efficacy of acalabrutinib, factors influencing acalabrutinib clearance and ACP-5862 formation and clearance were assessed. rCYP (recombinant cytochrome P450) reaction phenotyping indicated that CYP3A4 was responsible for ACP-5862 formation and metabolism. ACP-5862 formation K

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the beta 1c, beta 1i, beta 5c, and beta 5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting beta 2c or beta 2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the beta 2c- and beta 2i-selective-compounds LU-002c (4; IC50 beta 2c: 8 nM, IC50 beta 2i/beta 2c: 40-fold) and LU-002i (5; IC50 beta 2i: 220 nM, IC50 beta 2c/beta 2i: 45-fold), respectively. Co-crystal structures with beta 2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of beta 2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Background and Purpose Multicatalytic endopeptidase complex-like-1 (β2i), low molecular mass polypeptide (LMP) 2 (β1i) and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU-005i, a recently described inhibitor of the immunoproteasome. Experimental Approach The specificity of LU-005i and other immunoproteasome-selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin-stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model was assessed by measuring weight loss and colon length. Key Results LU-005i is the first human and mouse immunoproteasome-selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU-005i inhibited cytokine secretion from endotoxin-stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naive T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis. Conclusion and Implications This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.

Most known β2 selective proteasome inhibitor suffer from relatively poor cell permeability as the result of a net positive charge caused by the basic moiety at P1. Here we describe the synthesis of oligopeptide vinyl sulfones that contain different amino acids bearing amino groups with reduced basicity at P1 and/or P3. For this, we developed the first enantioselective synthesis of lysine(4-ene) and lysine(4-yn). These amino acids, as well as histidine and diaminopropionic acid-glycine, were incorporated at the P1 and/or P3 position of oligopeptide vinyl sulfones. All inhibitors inhibit β2, however, with loss of potency compared to our most potent and selective β2 inhibitor, LU-102. These results notwithstanding, our study described here provides important insights for the future design of β2 selective proteasome inhibitors.

Bruton tyrosine kinase (BTK) is an important target in oncology and (auto)immunity. Various BTK inhibitors have been approved or are currently in clinical development. A novel BTK inhibitor series was developed starting with a quinazoline core. Moving from a quinazoline to a quinoline core provided a handle for selectivity for BTK over EGFR and resulted in the identification of potent and selective BTK inhibitors with good potency in human whole blood assay. Furthermore, proof of concept of this series for BTK inhibition was shown in an in vivo mouse model using one of the compounds identified.

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. This paper describes a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes designed for β1i, β2i, β5c and β5i proved to be useful two-step ABPs that effectively label their designed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (IEDDA) ligation. The compound designed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro . The compound designed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide-alkyne click ligation chemistry both in vitro and in situ . In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which reporting on proteasome activities in living cells.

Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (β1c, β2c, β5c), the three homologous activities, β1i, β2i and β5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of β1i, β2i and β5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present.

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2, β5), while β5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of β1 or β2 with β5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective β2/β5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of β5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the β5/β2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.

Human proteasomes are validated targets in oncology and are promising targets in immune related diseases. A number of peptide-based proteasome inhibitors have reached the clinic and several others are currently investigated in clinical trials. Despite these achievements, peptide-based inhibitors may be susceptible to protease-mediated degradation. In this paper, we report on our results on the introduction of 5-methylpyridin-2-one moiety in peptide vinyl sulfone and peptide epoxy ketone-based proteasome inhibitors. Pyridin-2-ones have previously been used as constrained peptide isosteres capable to act as a hydrogen-bond acceptor. Our results demonstrate that 5-methylpyridin-2-one can be introduced into proteasome inhibitors, but that the activity of the resulting compounds is compromised compared to their parent peptide vinyl sulfones and peptide epoxy ketones.

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, β1c, β2c, and β5c. Lymphoid tissues also express the immunoproteasome subunits β1i, β2i, and β5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for β1c, β2c, β5c, and β2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of β5i and β1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.

In recent years, the field of covalent kinase drug discovery has been rapidly evolving. Several covalent kinase inhibitors (such as afatinib, osimertinib, ibrutinib and acalabrutinib) have obtained market approval or are currently in early and advanced clinical studies. Both covalent irreversible and covalent reversible kinase inhibitors have been discovered, providing an opportunity to target specific kinases, mutations and or pharmacokinetic (PK)/pharmacodynamic (PD) profiles. This chapter will give an overview of the state-of-the-art of covalent kinase drug discovery. First, the concept of covalent enzyme inhibition will be introduced and a comparison to non-covalent inhibitors will be made, highlighting possible advantages and disadvantages of such mechanisms of action (MOA). The importance of selectivity, drug-target residence times and inhibitor kinetics will also be discussed. Finally, a literature overview of irreversible and reversible covalent kinase inhibitors that are currently on the market or in the clinical testing phase will be presented.

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.

During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.

Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity.

Mammalian genomes encode seven catalytic proteasome subunits, namely, β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit β5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of β1c/β1i, β2c/β2i, or β5c/β5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable β1i and β5i selective inhibitors that outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.

Acalabrutinib is a potent and highly selective, oral covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma by the United States Food and Drug Administration in October 2017. ADME studies in humans, rat and dog, revealed extensive metabolism of acalabrutinib. The major circulating metabolite (M27, ACP-5862) was produced by CYP3A oxidation, resulting in a pyrrolidine ring-opened metabolite, with the butynamide electrophile still present. We investigated the on-target BTK inhibition and kinase selectivity profile of ACP-5862 using the same biochemical and cellular assays previously employed to profile acalabrutinib.1 The apparent BTK IC50 was determined over time using the LanthaScreen assay. Results indicate that ACP-5862, like acalabrutinib, is a covalent inhibitor of BTK. Binding kinetics derived from IC50 over time data, indicated that acalabrutinib and ACP-5862 have similar affinity (KI), however the BTK inactivation rate (kinact) for ACP-5862 was half that relative to acalabrutinib. This indicated a covalent BTK inhibition potency for ACP-5862 that was 2-fold lower than acalabrutinib. The overall kinome inhibition profile at 1 µM using KINOMEscan (Eurofins DiscoverX), as well as IC50 determinations on closely related kinases with a Cys in the same position as Cys481 in BTK (ITK, TXK, TEC, BMX, EGFR, ERBB2, ERBB4, BLK, JAK3), both revealed that the kinase selectivity profiles of acalabrutinib and ACP-5862 were similar. On-target inhibition in B cells was investigated measuring inhibition of B-cell antigen receptor-mediated activation of CD69 cell surface expression on human peripheral B cells using human peripheral blood mononuclear cells and human whole blood (hWB). The EC50 for anti-IgD-induced CD69 expression in hWB was 64 ± 6 nM for ACP-5862, compared to 9.2 ± 4.4 nM for acalabrutinib. The hWB EC90, representing near complete inhibition of BTK by acalabrutinib and ACP-5862 was observed at 72 ± 20 nM and 544 ± 376 nM, respectively. In conclusion, the results indicate that ACP-5862, the major metabolite of acalabrutinib, has intrinsic BTK inhibitory activity and a similar kinase selectivity profile as acalabrutinib. The contribution of ACP-5862 to on-target covalent inhibition of BTK in humans is unclear at present, but is most likely limited, since the EC90 in hWB for ACP-5862 approximated observed plasma Cmax in humans dosed with 100 mg acalabrutinib. The relative contribution of acalabrutinib and ACP-5862 to BTK pharmacodynamics is under further investigation. Terry Podoll and J. Greg Slatter are former Acerta Pharma employees. Reference 1. Barf T, Covey T, Izumi R, et al. Acalabrutinib (ACP-196): A covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J Pharmacol Exp Ther. 2017;363(2):240-252. Citation Format: Allard Kaptein, Terry Podoll, Gerjan de Bruin, Maaike Emmelot-van Hoek, Anouk de Jong, Bart van Lith, Niels Hoogenboom, Tjeerd Barf, Joseph Ware, J. Greg Slatter. Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2194.

The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

Inhibition of the immunoproteasome subunit β5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' β' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human β5c/β5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human β5c, human β5i or mouse β5i and the neighboring β6 subunit. The structural data reveal exceptional conformations for the most selective human β5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of β5i inhibitors with potential clinical applications.

This work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large hydrophobic P3 residue and small hydrophobic P1 residue, that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity relationships detail how in a logical order the β1c/i, β2c/i, and β5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers, and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed with some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and efficiently blocks β5c in cells expressing both constitutive proteasomes and immunoproteasomes.

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein: Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The incorporation of adamantylalanine and carboranylalanine at the P2 site of bortezomib is well tolerated and provided potent cell permeable proteasome inhibitors with increased off-rates compared to bortezomib. Adamantylalanine and carboranylalanine were synthesized enantioselectively by an asymmetric Strecker reaction on Ellmans tert-butyl sulfinimines.

Purpose Proteasome-inhibiting drugs (PI) are gaining importance in hematologic oncology. The proteasome carries three proteolytically active subunits (β1, β2, β5). All established PI (bortezomib and carfilzomib), as well as experimental drugs in the field (dalanzomib, oprozomib, and ixazomib), by design target the rate-limiting β5 subunit. It is unknown whether β2-selective proteasome inhibition can also be exploited toward anticancer treatment. Combining PI with the pan B-cell-directed Bruton tyrosine kinase inhibitor ibrutinib appears a natural option for future improved treatment of multiple myeloma (MM) and B-cell lymphomas. However, bortezomib induces phosphorylation of IκB and activation of NF-κB in MM cells, while ibrutinib inhibits the IκB/NF-κB axis, suggesting antagonistic signaling. A β2-selective proteasome inhibitor may lack such antagonistic signaling effects. Methods We recently introduced LU-102, the first β2-selective PI available for preclinical testing. We here compare bortezomib with carfilzomib and LU-102 in MM and MCL in vitro with regard to their effects on pIκB/NF-κB signaling and their cytotoxic activity in combination with ibrutinib. Results LU-102 reduced phosphorylation of IκB, in contrast to bortezomib and carfilzomib, and was a superior inhibitor of NF-κB activation in MM cells. This translated into highly synergistic cytotoxicity between LU-102 and ibrutinib, which was able to overcome BTZ resistance and CFZ resistance. By contrast, BTZ lacked consistent synergistic cytotoxicity with ibrutinib. Conclusion Ibrutinib is highly synergistic with β2-selective proteasome inhibition against MM and MCL in vitro. Novel β2-selective proteasome inhibitors may be exploited to overcome bortezomib/carfilzomib resistance and boost the activity of BTK inhibitors against B-cell-derived malignancies.

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',β'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. (3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. (4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.

A concise and practical preparation of the non-proteinogenic amino acid l -kynurenine is reported. The synthetic approach is scalable and provides ready access to this valuable amino acid in either l - or d -stereochemistry starting from l - or d -tryptophan, respectively. In the optimized procedure, two discreet oxidation steps are applied sequentially to convert the tryptophan indole ring into the keto-aniline moiety contained within the kynurenine side chain.

Item does not contain fulltext Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though with considerable loss of activity.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.

During an immune response to microbial infection, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt the terminal effector and memory fates. Here we show that ‘pre-effector’ and ‘pre-memory’ cells arising from the first CD8+ T cell division in vivo exhibit low and high rates of proteasome degradative activity, respectively. Pharmacologic inhibition of proteasome activity in activated CD8+ T cells resulted in increased inflammatory cytokine production and effector cell-associated molecule expression in vitro, as well as a loss of survival capacity and an impaired recall response to secondary infection in vivo. By contrast, increasing proteasome activity using a novel small molecule proteasome activator resulted in augmented memory cell-associated characteristics in vitro and an increased proportion of central memory cells with enhanced recall capabilities in vivo. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in activated CD8+ T cells affected processes associated with cellular metabolism. These proteasome-induced metabolic consequences were mediated, in part, through asymmetric segregation of Myc, a transcription factor that has been shown to control glycolysis and proliferation, during cell division. Taken together, these results suggest proteasome degradative activity as a key regulator of CD8+ T lymphocyte fate specification by virtue of effects on cellular metabolism.